Tumor imaging with cis-4-[18F]fluoro-L-proline (cis-FPro) was compared to that of L-[3H]proline and L-[3H]methionine in F98 rat gliomas by dual-tracer autoradiography. All tracers exhibited high accumulation in the tumors but in the normal brain significant uptake was observed for L-[3H]methionine only. Tumor extent on autoradiograms with L-[3H]proline and L-[3H]methionine was identical to that of histological staining while autoradiograms of cis-FPro showed diffuse uptake in the penumbra of some tumors. First PET studies in 7 patients with cerebral gliomas demonstrated accumulation of cis-FPro in tumor areas with enhancement of Gd-DTPA on MR scans. Uptake of cis-FPro in normal brain tissue was negligible. In one patient with a glioblastoma accumulation of cis-FPro was also found in two brain areas without enhancement of Gd-DTPA on MR scans. Control of MRI suggested tumor growth in these areas at further follow up. Our results indicate that in most gliomas increased cis-FPro uptake is restricted to areas with disruption of the BBB which limits its clinical utility.